Shewanella oneidensis MR-1 is composed of two highly homologous flagellins, FlaA, and the major structural unit, FlaB. We identified a gene cluster, SO_3261-SO_3265 (now sfmABCDE), that is required for the formation of a fully functional filament and for motility. The predicted function of the corresponding gene products strongly indicated a role in flagellin modification. Accordingly, loss of sfmABCDE results in a significant mass shift of both FlaA and FlaB. Mass spectroscopy analysis and single residue substitutions identified five serine residues in both flagellins that are modified via O-linkage. Modeling of the flagellin structures strongly suggests that at least four of the modified residues are exposed to the filament’s surface. However, none of the five serine residues solely is crucial for function and assembly. Structural analysis of the flagellin modification revealed that it likely contains a nonulosonic acid (274 Da) linked to each glycosylated serine. The putative nonulosonic acid is further substituted with a 236 Da moiety which can carry additional methyl groups . In addition, at least 5 lysine residues in FlaB and one in FlaA were found to be methylated. Based on homology comparisons we suggest that smfABCDE is required for species-specific flagellin modification in S. oneidensis MR-1.The unsheathed flagellar filament of  Aeromonas hydrophila, Bdellovibrio bacteriovorus, Campylobacter sp., Caulobacter crescentus, or Vibrio sp. possess flagella which are composed of several different flagellins For many bacteria, the capability to actively move towards more favorable conditions provides an important advantage for successful propagation and survival Vibrio sp., are sheathed in a membrane-like structure Campylobacter species. For this organism, pseudaminic acid (Pse) is among the predominant O-linked glycans, and the linkage occurs prior to the transport of the flagellin and assembly of the filament. The flagellin of Helicobacter pylori has been demonstrated to be similarly decorated with Pse A. hydrophila also strongly suggests a role of Pse in flagellin glycosylation of both polar and lateral flagellar systems The flagellar filaments of some species, such as the polar flagellum of Shewanella belong to the group of facultatively anaerobic gammaproteobacteria. Generally, all members of this genus exhibit a pronounced respiratory flexibility that allows them to use a wide range of alternative electron acceptors when growing under anaerobic conditions, among them numerous metal ions, including radionuclides, and halogenated compounds Shewanella species are thought to be suitable organisms for applications in bioremediation of contaminated soils or production of microbial fuel cells Shewanella are motile by at least one flagellar system, forming a single sodium ion-driven polar flagellum. For S. oneidensis MR-1, flagella-mediated motility allows a directed movement towards redox-active surfaces S. oneidensis has demonstrated the presence and differential regulation of two flagellins, FlaA and FlaB Shewanella and demonstrate a role of the protein in flagellin modification. However, the exact mechanism of flagellin modification in Shewanella and the factors involved therein are still mostly unknown. In this study, we identified a novel gene cluster whose gene products are involved in flagellin modification. We show that the major subunit FlaB is likely modified at five different surface-exposed serine residues, and we further characterized the modification.Species of the genus S. oneidensis MR-1 using a Tn5-based transposon system. The resulting mutants were screened for alterations in their ability to form biofilms in a static microtitre plate assay 28) consensus sequence (TAAAG-N14-GCCGTTAA) is predicted to be located 57 bp upstream of SO_3261 ilvB) is 1746 bp in length, the deduced protein of 581 amino acids has a predicted molecular mass of 63.0 kDa. The protein has a thiamine pyrophosphate (TPP)-binding domain and has been annotated as TPP-dependent enzyme involved in flagellin modification. The third gene in the operon, SO_3263, has a length of 768 bp and encodes a protein of 255 amino acids, which is predicted to be a 3-oxoacyl-(acyl-carrier-protein) reductase. The fourth gene in the operon, SO_3264, 1188 bp, is predicted to encode a protein of 395 amino acids (44.7 kDa) with a putative function as an S-adenosylmethionine (SAM)-dependent methyltransferase. Finally, the last gene in the operon, SO_3265  contains an ARP-grasp_4 domain, suggesting a carboxylate-amine ligase or peptide modification activity During the course of a previous study, a transposon mutagenesis was conducted on To elucidate the role of the genes SO_3261-3265 in flagella-mediated motility, we introduced defined in-frame deletions into the corresponding genes. We then determined the swimming ability of the resulting mutants in soft agar plates and under the light microscope. Single deletions of each of the first four genes of the operon (SO_3261-3264) resulted in strains that were non-motile on soft agar plates . Under tS. oneidensis MR-1 (sfmABCDE (for Shewanella flagellin modification).The striking motility phenotype of the mutants, the inability of the mutants to assemble a fully functional flagellar filament, and the putative functions of the proteins encoded by some of the genes in the operon strongly suggested a role in modification of the flagellin. We therefore determined whether loss of the genes encoded in the operon results in a significant mass shift of the flagellins. To this end, whole protein extracts obtained from the wild type and corresponding mutants where separated by polyacrylamide gel electrophoresis (PAGE), and the flagellins were detected subsequent to Western transfer using antibodies raised against the flagellar filaments of sis MR-1 . Both FlS. oneidensis MR-1 have provided evidence for a glycosylation of the flagellin protein, however, the corresponding sites as well as the nature of the modification remained unknown. To first analyze the chemical nature of the modification, we profiled the reduced, carboxyamidomethylated and trypsin-digested protein extract from S. oneidensis flagellin by various mass spectrometry techniques and analyzed the resulting data with Proteome Discoverer  using the Sequest algorism, searching the detected peptides against S. oneidensis flagellar sequences FlaA and FlaB. Both proteins share 89% identity and 93% similarity. In this way, we identified 67% of the peptides from FlaA and 82% of the peptides from FlaB. The results confirmed that both protein forms were present in the extract , were doubly charged suggested that the 274 portion carries a basic functional group which can be easily be protonated, such as an amino group. Neither the oxonium ion of the 264 Da moiety (expected at m/z 265), nor its de-methylated or dehydrated forms were detected in any glycopeptide carrying 510, 524 and 538 Da modifications, suggesting that the 264 Da moiety does not contain nitrogen. The MS/MS data from S168SLSVGSLGNTTSAAR183, A164SNKSSLSVGSLGNTTSAAR183, A164SNKTSLSVGALNNATSANR183 and T168SLSVGALNNATSANR183 peptides with excess masses above 538Da were very complex. This indicated that these tryptic peptides may contain multiple modification sites, which was later confirmed by LTQ-ETD-MS/MS analysis (described in the section below). Working with the assumption that there were two occupied sites, we were able to readily assign mass differences above 538 Da. For example, the 1048 Da mass excess was predicted as 510/538 Da combination and/or 524/524 Da combination, whilst two 538 Da increments gave 1076 Da, and these predictions were confirmed by LTQ-ETD-MS/MS analysis. For the four peptides, the only exception was the 1090 Da mass excess  for site mapping. ETD is a fragmentation method that effectively cleaves peptide bonds without breaking the bond between the modified amino acid and its modification, making this method ideally suited for site mapping experiments. We digested the flagellin protein with trypsin without removing the modifications and separated and analyzed the tryptic digest with an ETD-equipped LTQ-Orbitrap XL. ETD-MS/MS generated mainly c and z ions from peptide bond cleavages with minimum loss of the post-translational modifications, thus allowing us to determine the site of modification. flaB genes were integrated into the chromosome to replace the native copy of flaB. On soft agar plates, all mutants bearing a single serine to alanine substitution were able to swim, albeit at a decreased level when compared to that of the wild type. To determine whether the serine substitutions also result in a mass shift of the flagellin, as would be expected by a loss of modified residues, we separated crude protein extracts obtained from the wild type and corresponding mutants by PAGE. The position of the flagellins was then detected after Western transfer by immunostaining. Two distinct bands were detected when FlaB carried substitutions in S143, S171, S180, and S185 . The accordingly modified and S185 . The uppProteome Discoverer 1.0 with the search criteria allowing 510-, 524- and 538 modifications and methylations. The data analysis by the software strongly indicated that the methylation occurs at Lysine 167, in addition to the 538 Da modifications on Serines 171 and 180. The modified peptides, including sites of methylation detected in this study are summarized in S. oneidensis flagellar extract.Moreover, we analyzed ETD-MS/MS data for the mapping of the site of possible methylations on amino acids, indicated by our primary LC-MS data analysis described above. An ETD MS/MS spectrum of m/z 1000.12 (3+), A164SNK167@SSLS171@VGSLGNTTS180@AAR, which includes the site of methylation at lysine 167, is shown in S. oneidensis flagella by high resolution, high accuracy mass spectrometry to gain accurate precursor- and fragment mass of the unknown modification for a more in-depth structural characterization. We measured the precursor ions with the high accuracy LTQ Orbitrap-XL, featuring 60,000 resolution (FWHM at m/z 400) and detected the doubly charged ions of the modified peptides, L137LAGGFSAGK146 with a post translational modifications of 510, 524 or 538 Da, at m/z 715.8683, m/z 722.8751 and m/z 729.8838, respectively .We further analyzed tryptic peptides from ectively . The mas137LAGGFSAGK146 with the 538 Da modification. We detected mostly y and b ions from the peptide backbone, which were all identified within 1 ppm mass accuracy, but also detected ions that did not match with any predicted peptide backbone fragments. These were most likely oxonium ions and other fragment ions of the modification. The 274 Da portion of the modification was observed as an oxonium ion at m/z 275.1238. The most likely formula for this oxonium ion is C11H19N2O6+ . We also detected signals corresponding to one, two, and three losses of water from the 274-Da moiety with masses of 257.1133 Da , 239.1028 Da , and 221.0921 Da . We also detected an ion resulting from the loss of an acetyl group from the 274-Da precursor at 233.1134 Da  and three ions at 180.0654 Da , 162.0550 Da  and 134.0600 Da . A modification with a similar fragmentation pattern has been described in Campylobacter flagella and was identified as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (di-N-acetyl pseudaminic acid or Pse5Ac7Ac) S. oneidensis. The 42-Da difference corresponds to the lack of one N-acetyl group, and the theoretical mass of mono-N-acetyl pseudaminic acid agrees with our observed mass of m/z 275.1238 , whereas the 250 Da and the 236 Da moieties in the 524 Da and the 510 modifications were predicted to be C10H18O7 and C9H16O7, respectively .We furthermore performed highly accurate HCD-MS/MS analysis on the tryptic peptides to determine the molecular formula of each portion of the unknown modifications. HCD fragmentation is available for the LTQ Orbitrap 275.1238 ; Table 4275.1238  and S4. S. oneidensis flagellar protein in larger scale and purified tryptic peptides/glycopeptides from the sample by IT-TOF for further structural characterization of the modification by NMR. We reduced, carboxyamidometylated and digested with trypsin the resulting 8 mg of flagellar protein extract and separated the digest by LC-IT-TOF. We isolated the fraction containing mostly L137LAGGFSAGK146 with the 538-Da modification and analyzed it by NMR . In ETD fragmentation analysis of modified peptides with 538 Da and 524 Da, we observed a neutral loss of 32 Da from the precursor glycopeptides, corresponding to loss of one methanol (CH3OH). This indicated that these modifications contain at least one O-methyl group, which was confirmed by NMR. These data support our prediction that the 14 mass differences observed between the 510, 524 and 538 Da modifications are due to different methylation stages. Since the 274-Da moiety does not carry any O-methyl group, we hypothesize the O-methylation(s) occur at the residue attached to the 274-Da moiety.In summary, the 274 Da increment of the modification is most likely a nine carbon keto acid sugar (belonging to the nonulosonic acid family) containing two nitrogens and one N-acetyl group. This residue is located at the reducing end of the glycan and is attached to serine. By considering the previous findings from other groups regarding flagella modification, it is highly likely that this sugar is a member of the pseudaminic acid family. The chemical formulae of the 238-, 250- and 264-Da moieties were determined by mass spectrometry . OxoniumC. jejuniP. aeruginosa FliC S. oneidensis MR-1 is similarly modified at at least 4, more likely 5 serine residues in FlaB. The O-linkage occurs to a glycan of 274 Da that, according to our analysis, most likely is a pseudaminic acid with a single acetyl substitution. Such nonulosonic acid have previously been demonstrated to occur in flagellin modifications of, for example, Campylobacter sp. and H. pylori . In addition, SfmC, which is predicted to function as a 3-oxoacyl-ACP-reductase, shares weak homologies to PtmA of C. coli (25%/43%), which has been implicated to play a role in flagellin modification in this species sfm operon differ significantly from factors that have been so far described to be involved in flagellin modification. SmfD is predicted to function as a methyl transferase, which is consistent with our observation of mass increments corresponding to one or two methyl groups being attached to the 236 Da moiety. Loss of the smf genes results in a significant mass shift of the FlaA and FlaB flagellins, strongly indicating that the corresponding gene products are crucial for glycan formation, their linkage to the serine residues in FlaC, or both. In Campylobacter sp., the transfer of the glycan to the flagellin is thought to be mediated by PseE and/or PseD, which have been demonstrated not to be required for the glycan formation cascade smf operon, but likely not part of the operon. Similar homologies of SO_3259 occur to PseD (25%/40%). A second protein, SO_3273, also annotated as MAF, exhibits a similar degree of homology to PseE (22%/41%) and PseD (22%/35%). Thus, these two proteins might be another part of the S. oneidensis MR-1 flagellin glycosylation machinery and might function in glycan transfer to the flagellin. Notably, the mass shift of the flagellins in strains lacking SfmA-D appears to be similar, indicating that the modification needs to be fully assembled prior the transfer to the protein.Generally, O-glycosylation of the flagellins is thought to occur before the flagellins are secreted through the flagellar type III export apparatus S. oneidensis MR-1 FlaB the four serine residues are located between the highly conserved putative D0 and D1 regions, which are, based on the structure of Salmonalla FliC, predicted to be required for flagellin export and assembly S. oneidensis MR-1 FlaB using the Protein Homology/Analogy Recognition Enginge V 2.0 (Phyre2) Salmonella FliC, and that the serine residues of S. oneidensis FlaB, which were identified to be modified, are likely to be exposed to the surface of the filament  initiates with similar precursors such as pseudaminic (or a similar nonulosonic) acid, as described for other bacterial systems, while the final glycan modification occurs in a species-specific fashion. To further confirm the structure of the pseudaminic acid residue and to elucidate the structure of the 264-Da residue, we will remove the peptide by exhaustive protease digestion or β-elimination under harsher conditions. The purification of the glycan and its structural characterization are currently underway. Further analyses are also directed towards the question of whether the MAF proteins SO_3259 and SO_3273 are also part of the S. oneidensis glycosylation pathway. Our analysis also demonstrated that several lysine residues are methylated. So far, we have not determined whether or not function or if loss these methylations has any functional consequences. It is also unclear whether the sfm genes are involved in the observed lysine modifications. Thus, current studies address the question of the role of these additional modifications and whether the flagellins of other Shewanella species are similarly modified.Commonly, the modified peptides reside in the central region of the flagellin, the part of the protein which is thought to be located at the surface of the flagellar filament filament . One excs domain . The serS. oneidensis flagellins was published It should be noted that, while this manuscript was under review, another study on the posttranslational modification of the E. coli strains DH5α λpir and WM3064 were routinely carried out in LB medium at 37°C. Medium for the 2,6-diamino-pimelic acid (DAP)-auxotroph E. coli WM3064 was supplemented with DAP at a final concentration of 300 µM. S. oneidensis MR-1 was cultured in LB at 30°C. Solid media were prepared by adding 1.5% (w/v) agar. When appropriate, media were supplemented with 100 µg ml−1 ampicillin sodium salt, 50 µg ml−1 kanamycin and/or 10% (w/v) sucrose. For promoter induction of pGEX plasmids, IPTG was added to a final concentration of 1 mM in liquid medium.Bacterial strains and plasmids used in this study are summarized in S. oneidensis MR-1 following essentially the protocol that was described earlier E. coli strains by transformation using chemically competent cells flaB mutant versions into S. oneidensis MR-1 was performed using a kanamycin-resistance/sucrose-sensitivity counterselection procedure as described earlier flaB  were constructed by overlap extension PCR using mismatched primer pairs. Markerless in-frame deletion mutants of S. oneidensis MR-1 leaving only short terminal sections of the target genes were constructed basically as reported earlier, using the suicide vectors pGP704Sac28KM  for construction of sfmABCDE, and pNPTS138-R6KT flaAB, pilMNOPQ and mshA deletion plasmids. Primer sequences are available upon request. For an overexpression plasmid of the flaB N-terminal domain the corresponding fragment was amplified using appropriate primers prior to integration into pGEX-4T-1 . Overproduction of the protein and purification was carried out following the manufacturer’s instructions.DNA manipulations were performed using standard protocols or following the manufacturer’s instructions S. oneidensis MR-1 cells grown to the exponential phase under aerobic conditions in LB (OD600 of 0.5 to 0.7) were harvested by centrifugation at 4,600×g for 15 min at 4°C. Resulting cell pellets were immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted from S. oneidensis MR-1 cells by using the hot-phenol method For total RNA extraction of Motility assays were basically carried out on soft agar plates as described before pilM-Q ΔmshA) were incubated to the exponential phase and harvested at 13.000×g prior to resuspenision in TBS . Flagellar filaments were sheared off the cell bodies by harsh vortexing at 4°C for 1 hour. The filaments were subsequently separated from the cell bodies through slow centrifugation at 6000×g at 4°C, giving rise to a supernatant containing the soluble flagellar filaments. Ultra-centrifugation at 120,000×g was used to harvest and concentrate the flagellar filaments for subsequent procedures. Flagellar pellets were resuspended in TBS and frozen at −20°C for long-term storage.For preparation of native flagellar filaments and subsequent mass spectroscopic analyses a protocol was used similar to what was described before 4HCO3 and digested with Trypsin (Promega), at 37°C overnight. A small aliquot of the tryptic digest was profiled by LC-MS/MS and the rest of the digest was further digested with Glu-C (Roche) at 25°C overnight and dried by either a speed vacuum concentrator or lyophilization. The dried sample was resuspended with solvent A (0.1% formic acid in water) and passed through a 0.2 µm filter  prior to mass spec analysis.The sample was reduced with 25 mM dithiothreitol (DTT) for 1 hr at 55°C and carboxyamidomethylated with 90 mM iodoacetamide in the dark for 45 min. The sample was dialyzed with a 4 kDa cut-off membrane (Millipore) against nanopure water at 4°C overnight to remove salts and then dried in a Speed Vac. For the sample analysis/purification by LC-ETD/HCD-MS/MS and IT-TOF the sample was digested with Trypsin  for IT-TOF). For O-glycosylation site mapping by BEMAD Tryptic digests were analyzed by nano-LC-ESI-QTOF-MS/MS using a reverse-phase nano-HPLC system  connected to a quadrupole TOF mass spectrometer . The digests were separated by a binary nano-HPLC gradient generated by an Ultimate pump fitted with a Famos autosampler and a Switchos microcolumn switching module . An analytical C18 nanocapillary  and a micro precolumn C18 cartridge were employed for on-line peptide separation. The digest was first loaded onto the precolumn and eluted with 0.1% formic acid (Sigma) in water  for 4 min. The eluant was then transferred onto an analytical C18 nanocapillary HPLC column and eluted at a flow rate of 150 nL/min using the following gradient of solvent A [0.05% (v/v) formic acid in a 95∶5 (v/v) water/acetonitrile mixture] and solvent B [0.04% formic acid in a 95∶5 (v/v) acetonitrile/water mixture]: 99% A from 0 to 5 min, 99 to 90% A from 5 to 10 min, 90 to 60% A from 10 to 70 min, 60 to 50% A from 70 to 71 min, 50 to 5% A from 71 to 75 min, 5% A from 75 to 85 min, 5 to 95% A from 85 to 86 min, and 95% A from 86 to 90 min. Data acquisition was performed using Analyst QS software with an automatic information-dependent-acquisition (IDA) function.For the site of O-glycosylation mapping by BEMAD, LC-MS/MS analysis was performed on an LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. The filtered sample was loaded onto a nanospray tapered capillary column/emitter  self-packed with C18 reverse-phase resin  in a nitrogen pressure bomb at 1,000 psi and then separated via a 160 min gradient of increasing solvent B  at a flow rate of ∼500 nL/min directly into the mass spectrometer.S. oneidensis MR-1 protein sequences SO_3237 and SO_3238, using the TurboSequest algorithm. The SEQUEST parameters were set to allow 50.0 ppm of precursor ion mass tolerance and 0.8 Da of fragment ion tolerance with monoisotopic mass. Digested peptides were allowed with up to three missed internal cleavage sites, and the differential modifications of 57.0215 Da, 15.9949 Da and 136.0017 Da were allowed for alkylated cysteine, oxidation of methionines and DTT-modified serine and threonine, respectively. The peptides identified by the software were further filtered based on XCorr value The LTQ was run in automatic mode collecting an MS scan (Full FTMS at 300–2000 m/z) followed by data dependent MS/MS scans of either the six most intense ions from a calculated parent mass list or the six most abundant precursor ions if listed masses were not found. The parent masses were calculated from the tryptic, or trypsin/Glu-C peptides with potential O-glycosylation sites allowed for mass increase of 136.0017 for the DTT-modification. The resulting data were analyzed manually as well as by software . For the data analysis by the software, the data was searched against the In order to map and analyze post translational modifications, an LC-MS/MS analysis was performed on a ThermoFisher LTQ-Orbitrap XL equipped with a nanospray source and ETD and HCD fragmentation capabilities. The sample was loaded onto a C18 self-packed capillary column and separated by the same solvent and the same gradient program that was used for the BEMAD-derivative described above. An instrument method was used to collect a full mass spectrum, followed by fragmentation from a parent mass list or, if absent, the most abundant ions present in the spectra, via HCD and ETD. Dynamic exclusion was set at 1 for 30 seconds. Fourier Transform mode was used to fragment peptides utilizing 38% Higher Energy collision Dissociation (HCD) with a 2.5-m/z isolation width and a resolution of 15,000 (at 400 m/z). Ion Trap mode was used to fragment peptides utilizing Electron-Transfer Dissociation (ETD) for 100 ms with supplemental activation energy and a 3.0-m/z isolation width. The resulting data was analyzed manually as well as by the software  using the TurboSequest algorithm. For the analysis by the software, a precursor ion mass tolerance was allowed up to 10.0 ppm and fragment ion tolerance was set up to 0.8 Da with monoisotopic mass. Digested peptides were allowed with up to three missed internal cleavage sites, and the differential modifications of 57.0215 Da, 15.9949 Da and 14.0156 Da for alkylated cysteine, oxidation of methionines, and mwthylation respectively. Furthermore, the accurate delta mass of post translational modifications were calculated from the full mass data obtained in Fourier transform mode and used for making the parent mass list for selected MS/MS analysis and data analysis by the software.Purification of tryptic modified peptides was performed on an IT-TOF (Shimadzu). The tryptic digest was filtered, and 100 µL was injected via autosampler. Separation was accomplished by a C18 reversed-phase HPLC Column , and the sample was eluted by a gradient elution with solvent A (0.1% formic acid in water) and B  using a 70 min gradient of increasing solvent B at a flow rate of 0.7 mL/min. The eluent was split at a 1∶300 ratio. The smaller portion was measured by full mass analysis in positive ion mode, followed by MS/MS analysis of the six most abundant ions. The rest was monitored by PDA with the range of 190 nm–600 nm and then collected in a fraction collector. The fraction collector was set to collect 1 mL/fraction. The fractions containing modified peptide were dried in a speed vacuum concentrator and analyzed by NMR.2O , dissolved in 270 µL D2O , and placed into a 5-mm NMR tube with D2O-matched magnetic susceptibility plugs (Shigemi). One-dimensional proton and 2-D gradient-enhanced COSY NMR spectra were acquired at 25°C on a Varian Inova 800 spectrometer, equipped with a cryogenic triple-resonance probe. Spectral width was 7184 Hz, and 256 increments with 8 scans each were collected. Acquisition time was 150 ms, resulting in 1078 datapoints per FID, which were zero-filled to 2048 final points using the Mestre-C software. The data was processed with a sinebell function in both dimensions and linear prediction  in F1. Chemical shifts were referenced to internal acetone (δ = 2.218 ppm).The isolated modified peptides were deuterium-exchanged by lyophilization from DStaining of flagellar filaments was carried out as described earlier 600 of 10. Subsequently, samples were heated at 99°C for 5 min, and stored at −20°C. 10 µl of sample were resolved by SDS-PAGE using 15% polyacrylamide gels. Proteins were transferred to nitrocellulose Immobilon-P membrane  by semidry transfer. For detection of flagellin FlaA and FlaB, polyclonal antibodies were used that were raised against the N-terminal conserved region of FlaB  in the dilution of 1∶600. Secondary anti-rabbit immunoglobulin G-horseradish peroxidase antibody was used at a dilution of 1∶20 000 for detection of N-terminus FlaB antibodies. Signals were detected using the SuperSignal® West Pico Chemiluminescent Substrate  and documented using the CCD System LAS_4000 .To determine the molecular mass of flagellin protein, lysates from exponentially growing LB cultures were obtained for Western blot analyses. Appropriate cells were harvested by centrifugation and resuspended in sample buffer Figure S1RT PCR analysis of the putative sfm operon start. The upper panel displays the genetic organization of sfmABC and the upstream genes SO_3260 and SO_3259. The positions of the expected PCR products obtained from cDNA are indicated above. Lower panel: RT PCR analysis. Lane I: control with chromosomal DNA as template; lane II, cDNA template; lane III, control assay without Reverse Transcriptase; lane IV, no template.(PDF)Click here for additional data file.Figure S2Protein identification in S. oneidensis flagellar extract by mass spectrometry. The flagella extract of S. oneidensis was reduced, carboxyamidomethylated and digested with trypsin, and profiled by various mass spectrometry techniques. The figure represents the data from LTQ-orbitrap-XL (LC-NSI-CID-MS/MS). The resulting data was analyzed by Proteome Discoverer  using the Sequest algorism, and the detected peptides were searched against S. oneidensis flagella sequences SO_3237 (FlaB) and SO_3238 (FlaA). The peptides identified by the software are highlighted in green. 82% of the peptides from SO_3237 (FlaB) and 67% of the peptides from SO_3238 (FlaA) were identified from the protein extract.(PDF)Click here for additional data file.Figure S3137LAGGFS@AGK146 plus a 522 Da modification.HCD-MS/MS spectrum from L (@represents the site of modification). The HCD-MS/MS spectrum showed accurate mass of glycan-related ions correspond to dehydrated ions of the 522 Da modification, and oxonium ion from the 274 Da moiety of the 522 Da modification (A). The series of fragments from the 274 moiety were observed in the mass range between 100-300 m/z (B). An inset in the spectrum C shows a proposed structure of the 274 Da moiety (observed m/z of 274 Da moiety as an oxonium ion is m/z 275). Pentagon cartoon and grayed circle cartoons in the figure represents 274 Da- and 250 modifications, respectively.(PDF)Click here for additional data file.Figure S4137LTGTFS@AGK146 plus a 510 Da modificationHCD-MS/MS spectrum from L (@represents the site of modification). The HCD-MS/MS spectrum showed accurate mass of glycan-related ions correspond to dehydrated ions of the 510 Da modification, and oxonium ion from the 274 Da moiety of the 510 Da modification (A). (PDF)Click here for additional data file.Figure S5Glycopeptide purification by IT-TOF. The tryptic digest of S. oneidensis flagellar protein was prepared in larger scale and tryptic peptides/glycopeptides were purified from the sample by IT-TOF for further structural characterization of the modification by NMR. The sample elution was monitored on-line by IT-TOF mass spectrometry. The figure represents the spectra of the fraction 15, containing mostly L137LAGGFSAGK146 with the 538-Da modification, which was analyzed by NMR.(PDF)Click here for additional data file.Figure S62D-NMR analysis of a glycopeptide fraction purified from S.oneidensis. Two-Dimensional gradient-enhanced COSY NMR spectrum of Fraction 15, containing mostly L137LAGGFSAGK146 with the 538-Da modification, run at 800 MHz and 25 °C. The dotted lines connect the two H-3 protons and the H-4 proton of the nonulosonic acid detected in MS.(PDF)Click here for additional data file.Figure S7Alignment of the variable regions of Shewanella flagellins. Variable regions of S. oneidensis MR-1 FlaA and FlaB (aa 101-189) were aligned to corresponding flagellin sequences of Shewanella spp. harbouring flagellins with a similar domain structure (comprising between 260 and 280 aa). Modified residues in FlaB are marked with arrows and in red. At some positions, several flagellins have a threonine instead of a serine residue (marked in green) that might be similarly modified. Invariable residues are highlighted in yellow, and positions with a single amino acid variation are marked in grey. Locus tag coding: Swoo, S. woodyi; Ssed, S. sediminis; Shew, S. loihica; Shal, S. halifaxensis; Sden, S. denitrificans; Sputw3181, S. sp. W3-18-1; Shewana3, S. sp. ANA-3; Sbal183, S. baltica OS183; Shew185, S. baltica OS185; Sput200, S. putrefaciens 200.(PDF)Click here for additional data file.


Single nucleotide polymorphisms (SNPs) in microRNA-coding genes may participate in the process of carcinogenesis by altering the expression of tumor-related microRNAs. It has been suggested that two common SNPs rs2910164 in miR-146a and rs11614913 in miR-196a2 are associated with susceptibility to hepatocellular carcinoma (HCC). However, published results are inconsistent and inconclusive. In the present study, we performed a meta-analysis to systematically summarize the possible association between the two SNPs and the risk for HCC.We conducted a search of case-control studies on the associations of SNPs rs2910164 and/or rs11614913 with susceptibility to HCC in PubMed, EMBASE, ISI Web of Science, Cochrane Central Register of Controlled Trials, ScienceDirect, Wiley Online Library and Chinese National Knowledge Infrastructure databases. Data from eligible studies were extracted for meta-analysis. HCC risk associated with the two polymorphisms was estimated by pooled odds ratios (ORs) and 95% confidence intervals (95% CIs). 5 studies on rs2910164 and 4 studies on rs11614913 were included in our meta-analysis. Our results showed that neither allele frequency nor genotype distribution of the two polymorphisms was associated with risk for HCC in all genetic models. Similarly, subgroup analysis in Chinese population showed no association between the two SNPs and the susceptibility to HCC.This meta-analysis suggests that two common SNPs rs2910164 and rs11614913 are not associated with the risk of HCC. Well-designed studies with larger sample size and more ethnic groups are required to further validate the results. Primary liver cancer is the sixth most common cancer worldwide . Due to its poor prognosis and high fatality rates, the incidence and mortality rates are almost equal. Primary liver cancer causes 695,900 deaths every year, making it the third most common cause of cancer-related deaths MicroRNAs (miRNAs) are small non-coding, single-stranded RNA molecules with typical length of ∼22 nucleotides. Functioning as post-transcriptional regulators, miRNAs complementarily bind to target mRNAs and negatively regulate their stability and transcriptional efficiency Single nucleotide polymorphisms (SNPs) in miRNA-coding genes may have effects on either the expression or the function of miRNAs by altering the secondary structure of miRNA precursors, consequently leading to the aberrant expression of a series of target genes and contributing to cancer susceptibility We carried out a publication search in PubMed, EMBASE, ISI Web of Science, Cochrane Central Register of Controlled Trials, ScienceDirect, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI) databases with the following search terms: (miR-146a OR miR-196a2 OR rs2910164 OR rs11614913) AND (hepatocellular carcinoma OR liver cancer OR hepatoma), by two independent investigators . Publication date and publication language were not restricted in our search. Reference lists were examined manually to further identify potentially relevant studies. We contacted the corresponding authors of conference abstracts without sufficient data for additional information by e-mail. If no reply or the author refused to provide the data required in this meta-analysis, the study was excluded. All studies matching the inclusion criteria were retrieved for further examination and data extraction. Investigators include experts in hepatobiliary surgery, biologists and qualified graduate researchers. All of the investigators have received training in literature search, statistics and evidence-based medicine.Studies included in our meta-analysis had to meet all the following criteria: (1)evaluated the associations between the SNPs miR-146a rs2910164 and/or miR-196a2 rs11614913 and susceptibility to HCC, (2) studied on human beings, (3)in a case-control design, (4)detailed genotype data were provided for the calculation of odds ratio (OR) and 95% confidence interval (95% CI), (5)if serial studies of the same population from the same group were reported, the latest study was included. We assessed the methodological qualities of included studies by the description of study population, detailed genotyping methods, the set of controls and cases and related statistical methods. Included studies had to meet the following criteria regarding methodology: (1) participants recruited from comparable populations with similar demographic background in control and case groups, (2) proper diagnostic criteria to determine the disease status, (3) Hardy-Weinberg equilibrium (HWE) reached in control population, (4) proper method to determine the genotypes with detailed description of method used, (5) appropriate statistical analysis performed, (6) detailed allele and genotype frequencies reported.Two investigators (Zhongxia Wang and Yin Cao) independently extracted data from the included studies. Data extracted from eligible studies included the first author’s name, publication date, country origin, ethnicity, genotyping method, total numbers of cases and controls and genotype frequencies of cases and controls. The two investigators checked the data extraction results and reached consensus on all of the data extracted. If different results were generated, they would check the data and have a discussion to come to an agreement. Three senior investigators  were invited to the discussion if disagreement still existed.P<0.05 was considered representative of a departure from HWE. ORs and 95% CIs were calculated to assess the strength of the association between miR-146a rs2910164 or miR-196a2 rs11614913 polymorphism and susceptibility to HCC. Pooled ORs were calculated for allele frequency comparison , additive model , dominant model , and recessive model , respectively. The significance of pooled ORs was determined by Z-test and P<0.05 was considered as statistically significant. Statistical heterogeneity among the studies was checked by chi-square-based Q-test. A P-value greater than 0.10 for Q-test indicates no significant heterogeneity existed among studies P value of Egger’s test less than 0.05 was considered representative of statistically significant publication bias For each study, HWE was evaluated using the goodness-of-fit chi-square test. et al. A total of 367 articles were retrieved after first search in PubMed, EMBASE, ISI Web of Science, Cochrane Central Register of Controlled Trials, ScienceDirect, Wiley Online Library and CNKI databases. As shown in Q-test in all of the genetic models showed no significant heterogeneity. Therefore, the pooled ORs were calculated using fixed-effects model. No significant association between miR-146a rs2910164 polymorphism and susceptibility to HCC was identified in any of the genetic models .The association between miR-146a rs2910164 polymorphism and susceptibility to HCC was analyzed in five independent studies with 1,912 cases and 2,149 controls. Results of the meta-analysis are shown in P = 0.088; GC versus GG: OR = 0.911, 95% CI 0.754–1.101, P = 0.336; CC versus GG: OR = 0.845, 95% CI 0.693–1.029, P = 0.095; GC/CC versus GG: OR = 0.883, 95% CI 0.739–1.056, P = 0.173; CC versus GC/GG: OR = 0.904, 95% CI 0.786–1.039, P = 0.155)  .P = 0.182; TC versus TT: OR = 1.121, 95% CI 0.855–1.470, P = 0.408; CC versus TT: OR = 1.318, 95% CI 0.881–1.974, P = 0.180; TC/CC versus TT: OR = 1.200, 95% CI 0.874–1.647, P = 0.260; CC versus TC/TT: OR = 1.132, 95% CI 0.965–1.326, P = 0.127). The results are summarized in 4 studies involving 1,790 cases and 1,635 controls were assessed for the association between miR-196a2 rs11614913 polymorphism and HCC risk. Significant statistical heterogeneity was identified in the comparison of allele frequency, TC versus TT, CC versus TT and in the dominant model so that random-effects model was used in these models. Fixed-effects model was used in the recessive model. However, for rs11614913, none of the genetic models produced significant association between rs11614913 polymorphism and HCC risk .P>0.05).Funnel plot and Egger’s test were performed to assess the publication bias of the literature. Symmetrical funnel plots were obtained in both of the SNPs tested in all of the models. Egger’s test further confirmed the absence of publication bias in this meta-analysis  to 0.709 , indicating no significant difference of susceptibility to HCC between CC and GG genotypes. Similarly, synthesis of the data with HBV positive control population obtained a pooled OR without statistical significance . The consistency of the stratified analysis suggests that HBV infection status in control populations may not be the factor that affected the stability of the comparison between CC and GG genotypes. Caution should be made when interpreting the result of the comparison between CC and GG genotype.Every single study involved in this meta-analysis was deleted each time to examine the influence of the individual data set to the pooled ORs. For rs2910164, the study from Zhang P = 0.01), indicatThe research on miRNAs has led to novel insights into the molecular mechanism of HCC miR-146a rs2910164 is located in the stem region opposite to the mature miR-146a sequence. This G>C polymorphism results in a change from G:U pair to C:U mismatch in the stem structure of miR-146a precursor. Several case-control studies have investigated the association between miR-146a rs2910164 polymorphism and risks of various cancers.et al.P = 0.10) to 0.71 . Zhang et al’s study with a relatively large sample size may be the source of potential heterogeneity or bias in the comparison between genotype CC and GG and could cause a major influence on the estimation of the association between rs2910164 polymorphism and HCC risk. Since HBV infection is a well-established risk factor for the development of HCC In this meta-analysis, a total of 5 case-control studies were analyzed to provide a comprehensive assessment of the association between miR-146a rs2910164 polymorphism and HCC. Our results did not support a genetic association between rs2910164 and susceptibility to HCC. Neither allele frequency nor genotype distribution was significantly associated with susceptibility to HCC. Since the incidence of gene polymorphisms may vary between different ethnic groups and this variation may interfere with the detection of minor effect of SNPs on HCC risk, subgroup analysis in Chinese population was performed to further explore the potential association between rs2910164 and the risk of HCC. However, even within the same ethnic group, no association of statistical significance was observed. Sensitivity analysis showed that the study from Zhang Another SNP shown to have potential relationship to the risk for HCC is rs11614913 in miR-196a2. It is reported that C allele of rs11614913 increased the expression of mature miR-196a2 in HCC tissues 4 studies on the relationship between rs11614913 and susceptibility to HCC were included in our meta-analysis. To our surprise, we failed to find any association between rs11614913 polymorphism and the risk for HCC in any of the examined genetic models. Similarly, subgroup analysis in Chinese population also showed no association of miR-196a2 rs11614913 polymorphism with susceptibility to HCC. Sensitivity analysis found no significant influence of any single study on pooled ORs, indicating the stability of this meta-analysis was acceptable.Several previous meta-analyses systematically reviewed the potential association of the two common polymorphisms rs2910164 and rs11614913 with susceptibility to cancer et al.To the best of our knowledge, this is the first meta-analysis evaluating the potential association between two common polymorphisms rs2910164 in miR-146a and rs11614913 in miR-196a2 and susceptibility to hepatocellular carcinoma. However, caution should be made when interpreting the results due to some limitations of this meta-analysis. Firstly, heterogeneity was detected in some comparisons of rs11614913 and sensitivity analysis showed that the study from Zhang In summary, despite the limitation, this meta-analysis suggests that miR-146a rs2910164 and miR-196a2 rs11614913 polymorphisms may not be associated with the risk of HCC. Well-designed studies with larger sample size and more ethnic groups should be considered to further clarify the association. Moreover, other factors such as gender, age, HBV/HCV infection status and alcohol consumption should also be considered in future studies.Table S1MOOSE checklist.(DOC)Click here for additional data file.


Streptococcus suis is a major swine pathogen worldwide that causes meningitis, septicemia, arthritis, and endocarditis. Using animal models, a surface-associated subtilisin-like protease (SspA) has recently been shown to be an important virulence factor for S. suis. In this study, we hypothesized that the S. suis SspA subtilisin-like protease may modulate cytokine secretion by macrophages thus contributing to the pathogenic process of meningitis.S. suis deficient in SspA expression.Phorbol 12-myristate 13-acetate-differentiated U937 macrophages were stimulated with recombinant SspA prior to monitor cytokine secretion by ELISA. Our results indicated that the recombinant SspA was able to dose-dependently induce IL-1β, IL-6, TNF-α, CXCL8 and CCL5 secretion in macrophages. The heat-inactivated protease was still able to induce cytokine secretion suggesting a non-proteolytic mechanism of macrophage activation. Using specific kinase inhibitors, evidence were bought that cytokine secretion by macrophages stimulated with the recombinant SspA involves the mitogen-activated protein kinase signal transduction pathway. While stimulation of macrophages with low concentrations of recombinant SspA was associated to secretion of high amounts of CCL5, the use of recombinant SspA at a high concentration resulted in low amounts of CCL5 detected in the conditioned medium. This was found to be associated with a proteolytic degradation of CCL5 by SspA. The ability of SspA to induce cytokine secretion in macrophages was confirmed using a mutant of S. suis SspA may promote central nervous system inflammation associated with meningitis.In conclusion, this study identified a new mechanism by which the  Streptococcus suis is a major swine pathogen worldwide that causes meningitis, septicemia, arthritis, and endocarditis , UO126  and JNK inhibitor II [c-JUN N-terminal kinase (JNK) inhibitor], were evaluated for their effect on IL-6, CXCL8, and CCL5 secretion by macrophages.All treatments and cytokine determination were performed in triplicate and the means ± standard derivations were calculated. Differences were analyzed for statistical significance using the Student's t-test and were considered significant at P < 0.01.S. suis to induce an inflammatory response in PMA-differentiated U937 macrophages, its effect on cell viability was evaluated. The MTT test revealed that macrophage viability was not significantly reduced (less than 20%) by a treatment with the recombinant SspA at a concentration of up to 33 μg/ml. As reported in Figure Prior to determine the capacity of the recombinant SspA of S. suis was inactivated by heat treatment (30 min at 100°C). However, stimulation of macrophages with the heat-inactivated SspA was associated with a significantly higher amount of CCL5 in the conditioned culture medium compared to the treatment with the active recombinant SspA (72409 ± 848 versus 2370 ± 61 pg/ml). Lastly, the presence of polymyxin B during stimulation of macrophages with the recombinant SspA protease had no significant effect on the levels of cytokine produced. The efficacy of polymyxin B (1 μg/ml) in neutralizing the inflammatory activity of Escherichia coli LPS was demonstrated in preliminary assays.The effect of stimulating macrophages with heat-inactivated recombinant SspA or with active SspA in the presence of polymyxin  on the secretion of IL-6, CXCL8 and CCL5, the three cytokines produced in higher amounts by macrophages, was then tested. As reported in Table S. suis P1/7 or G6G at MOI of 100. As reported in Table To further support the inflammatory property of the recombinant SspA, we compared the SspA-deficient mutant G6G and the parental strain for their capacity to induce of IL-1β, TNF-α, IL-6, CXCL8 and CCL5 secretion in macrophages. The MTT test revealed that macrophage viability was not significantly reduced (less than 10%) by a treatment with cells of Lastly we investigated the capacity of the SspA protease to degrade CCL5, IL-6 and CXCL8, the tree cytokines produced in higher amounts by macrophages stimulated with the recombinant SspA. Recombinant cytokines were incubated with the SspA protease at concentrations ranging from 0.26 to 16.5 μg/ml and after 4 h, residual cytokines were determined by ELISA  or through a non-proteolytic mechanism, involving the mitogen-activated protein kinases (MAPK) ,31. SeveS. suis -39. In arophages  and fibrrophages .S. suis to avoid and delay neutrophil attraction and activation. Cytokine degradation by proteases is a phenomenon well described in group A streptococci. Sumby et al., reported the ability of Streptococcus pyogenes SpyCEP to reduce neutrophil activity though cleavage and inactivation of the human chemokine granulocyte chemotactic protein 2 (GCP-2) [S. pyogenes was reported to cleave CXCL8 [Streptococcus agalactiae CspA, inactivates the CXC chemokines GRO-alpha, GRO-beta, GRO-gamma, neutrophil-activating peptide 2 (NAP-2), and GCP-2 [Finegoldia magna, that shares many properties with the SspA of S. suis, has been shown to degrade the chemokine MIG/CXCL9 [S. suis has been previously reported [S. suis [Our data showed that the amounts of CCL5 in the conditioned medium of macrophages stimulated with the heat-inactivated recombinant SspA was higher compared to that detected following stimulation with the active SspA. This suggests that SspA may degrade this cytokine. Using ELISA, we clearly showed the capacity of the recombinant SspA to degrade dose-dependently CCL5. Since CCL5 possesses chemotactic activity for immune cells, its inactivation by the SspA may allow  (GCP-2) . In addive CXCL8 ,43. Morend GCP-2 . Lastly,IG/CXCL9 . Degradareported . The pro[S. suis .S. suis deficient in SspA expression. The secretion of IL-1β, TNF-α, and IL-6 was significantly less important when macrophages were stimulated with cells of SspA mutant compared to the stimulation with the parental strain. This strongly supports the contribution of SspA in S. suis induced inflammatory response in macrophages. On the other hand, CCL5 secretion was found to be higher following stimulation with the SspA-deficient mutant compared to the parental strain. This result supports the capacity of the recombinant SspA protease to degrade CCL5. The fact that no decrease in CXCL8 secretion was observed following stimulation of macrophages with the SspA-deficient mutant suggests that other cell surface components of S. suis, such as the cell wall [The ability of SspA to induce cytokine secretion in macrophages was confirmed using a mutant of ell wall , are likS. suis may modulate the inflammation state associated with meningitis. It may either induce the secretion of important pro-inflammatory cytokines or, when present at high concentration, cause the degradation of selected cytokines, such as CCL5 and IL-6.In conclusion, this study bought evidence that the subtilisin-like protease SspA of LB performed all the experimental work and prepared the first draft of the manuscript. DG conceived the study design and prepared the final version of the manuscript. All authors read and approved the final manuscript.


ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity.Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase  and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site.High resolution melting (HRM) analysis and sequencing of the entire Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability. ETFA; OMIM 608053, ETFB; OMIM 130410) or the electron transfer flavoprotein dehydrogenase  genes. However, in some patients, the disorder may result from a few, currently unidentified, disruptions to riboflavin metabolism [ETF or ETFDH, the acyl-CoA dehydrogenases are unable to transfer the electrons generated by the dehydrogenation reactions. This results in the accumulation of various intramitochondrial acyl-CoA esters. Recently, ETFDH mutations were reported to be major causes of riboflavin-responsive MADD [Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as glutaric aciduria type II, is an autosomal recessively inherited disorder of fatty acid metabolism . In the ETF and/or ETFDH genes for the purpose of achieving the final diagnosis of MADD. Previously, we reconfirmed the high prevalence of the c.250G > A (p.Ala84Thr) mutation in Taiwanese patients with riboflavin-responsive MADD, as well as in the normal population using a high-resolution melting (HRM) analysis [ETFDH gene was detected in 49 patients with homozygous mutations and 20 patients with heterozygous mutations. To date, this mutation has never been reported in Western countries [ETFDH gene. Currently, there is no related study to explore the underlying mechanism for the most common mutation (p.Ala84Thr).Characteristics such as organic aciduria may be detected only during periods of illness or catabolic stress. In the majority of cases, depending on the metabolic status of the patient, plasma acylcarnitine analysis may not provide a definitive diagnosis. Amongst the most daunting challenges facing MADD is metabolic heterogeneity, in particular, for the diagnosis of MADD. Thus, numerous molecular diagnostic methods have been established for analyzing the analysis . It shouanalysis . Intriguanalysis -11. A toETFDH gene) which constitutes the electron-transport pathway for a number of mitochondrial flavoprotein dehydrogenases that are mainly involved in fatty-acid and amino-acid metabolism. Riboflavin (the precursor of FAD) treatment has been shown to strikingly ameliorate the symptoms and metabolic profiles in MADD patients [Flavin adenine dinucleotide (FAD) is a cofactor for the electron transfer flavoprotein:ubiquinone oxidoreductase  of Kaohsiung Medical University Hospital.® Blood Kit (Macherey-Nagel), according to the manufacturer's instructions.The blood samples were collected from the patient and the family members as well as from normal controls with informed consents. The genomic DNA samples were extracted from peripheral whole blood using the NucleoSpinETFDH, as previously described [® 480 High-resolution Melting Master  1 × buffer which contained Taq polymerase, nucleotides, the dye ResoLight and 30 ng DNA. The primers and MgCl2 were used at a concentration of 0.25 μM and 2.5 mM, respectively for identifying the ETFDH gene mutations.HRM analysis was applied for screening the mutation of escribed . PCR was® 480 instrument (Roche Diagnostics) provided with the software LightCycler® 480 Gene-Scanning Software Version 1.5 (Roche Diagnostics).The HRM assays were conducted using the LightCyclerThe PCR program required a SYBR Green I filter (533 nm). The program consisted of an initial denaturation-activation step at 95°C for 10 min, followed by a 45-cycle program. The 45-cycle program consisted of the following steps: denaturation at 95°C for 15 s, annealing at 58°C or 60°C for 15 s and elongation at 72°C for 15 s with the reading of the fluorescence. The acquisition mode was single. The melting program consisted of three steps: denaturation at 95°C for 1 min, renaturation at 40°C for 1 min, and the subsequent melting that included continuous fluorescent readings from 60 to 90°C at a rate of 25 acquisitions per degree centigrade.To confirm the results of the HRM analysis, a sequencing analysis was also performed for all the samples. After the HRM analysis, the samples were purified using the FavorPrep™ PCR clean-up mini kit. The PCR products generated after HRM were sequenced directly. The sequence reaction was performed in a final volume of 10 μL, comprising 5 μL of the purified PCR product, 2.5 μM of each PCR primer and 1 μL of the ABI PRISM terminator cycle sequencing kit v1.1 (Applied Biosystems). The sequencing program started from 96°C for 1 min and then followed by 25-cycle PCR program. The program consisted of the following steps: denaturation at 96°C for 10 s; annealing at 50°C for 5 s; and elongation at 60°C for 4 min. The sequence detection was performed using the ABI Prism 3130 Genetic Analyzer (Applied Biosystems). To ensure that the mutation found in the patient was the one specifically present in patients with MADD, 60 healthy control chromosomes were also screened.SIFT tool -16 gener2 server [2 automatically uses an effective consensus strategy. The above mentioned strategy combines structural- [2 server selected the porcine ETF:QO (PDB entry: 2GMH) [Three-dimensional (3D) structures of the human ETF:QO were predicted using the (PS)2 server ,18. (PS)uctural- -21 compay: 2GMH)  as the my: 2GMH) . The modMolecular dynamics (MD) simulations were performed using the software GROMACS ver. 4.5.1 . OPLS-AAThe objective was to understand the structural and functional implications of the amino acid substitution. Thus, the trajectories of WT and MT were analyzed for the following structural properties as a function of time: a) the root mean square deviation (RMSD) of the Cα atoms with respect to the starting conformation; b) RMSD of the FAD-binding motif with respect to the starting conformation; and c) the B-factors  of the CNormal mode analysis (NMA) is a powerful tool to estimate the dynamics based on structure -31. Low-ETFDH gene revealed a novel T > C point mutation at position 383. The point mutations were confirmed by direct DNA sequencing . The sibling was a carrier of c.383T > C (p.Phe128Ser). The father and mother were carriers of c.383T > C (p.Phe128Ser) in exon 3 and c.250G > A (p.Ala84Thr) in exon 3, respectively  as the template  when compared with the WT values Figure  and 4C. The correlation map for ETF:QO is shown in Figure In this study, we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO using MD simulations and NMA. Interestingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site.8hxhE(D), is part of the Rossmann fold [In the FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif . A subseann fold ,40-42. HThe binding of FAD is important for the catalytic activity of flavoproteins as well as for the correct folding, assembly and protein stability -45. A reet al. [ETFDH mutations, p.Leu127Arg, p.Asp130Val, and p.Trp131Cys in riboflavin-responsive MADD patients in the north of China. Er et al. [et al. [ETFDH mutation, p.Leu127His, in riboflavin-responsive MADD patients in the Taiwanese population. Law et al. [ETFDH mutation, p.Pro137Ser, in a Chinese family with riboflavin-responsive MADD. In addition, Goodman et al. [ETFDH mutations, p.Arg175Leu and p.Arg175His [ETFDH mutation in exon 3, 4 and 5 be initially screened. However, based on the B-factors analysis, the atomic fluctuations of the p.Leu127Arg, and p.Arg175His were not significant at the β1/α1 loop, which was involved in FAD binding  and medium-chain acyl-CoA dehydrogenase (MCAD) proteins ,47. In aet al.  recentlyr et al.  and Lian [et al.  reportedw et al.  also repn et al.  reportedrg175His -8,50, weThe present study is the first demonstration of p.Ala84Thr and p.Phe128Ser by molecular modeling, MD simulations, and correlated motion analysis. Along with the obtained results of the MD simulations and NMA, the present findings strongly suggest that p.Ala84Thr and p.Phe128Ser are likely to alter the ETF:QO protein structure near the FAD-binding site as well as disrupt the stability of FAD binding, which is essential for the activation of ETF:QO. The change of hydrophobic interaction may destabilize FAD binding, giving rise to a less stable or inactive conformation. Overall, the obtained findings have implications for the pathogenic mechanism of riboflavin-responsive MADD.The authors declare that they have no competing interests.Concept and design of the experiments: TKE, CCC, YYL, HCC, JGC, JKH and YJJ. Performance of the experiments: TKE and CCC. Data analysis and discussion: TKE, CCC, YYL, HCC, YHC, JGC, JKH and YJJ. Contribution of reagents/materials/analysis tools: HCC, YHC, JGC and JKH. Manuscript preparation: TKE, CCC, JGC, JKH and YJJ. All authors read and approved the final manuscript.Table S1. Dried blood spot acylcarnitine profile measured using tandem mass spectrometry in the patient before and after riboflavin treatment.Click here for fileTable S2. The highlighted amino acid alanine (A) and phenylalanine (F) shown in yellow and pink, respectively, at positions 84 and 128 are conserved in all the orthologs.Click here for fileFigure S1. RMSD plots of the whole WT and MT (A) p.A84T and (B) p.F128S structures with respect to the starting conformation during the course of the MD simulations.Click here for fileFigure S2. Structure of ETF:QO with the positions of the identified amino acid changes near residue F128 in riboflavin-responsive MADD patients labeled.Click here for fileFigure S3. Structures of the WT and MT ETF:QO are drawn in cartoon putty representation. The red arrows indicate the positions of the mutations.Click here for file


To investigate the biological radiation dose-response for patients of limited-stage small-cell lung cancer (LS-SCLC) treated with high radiation dose.Two hundred and five patients of LS-SCLC treated with sequential chemotherapy and thoracic radiotherapy with involved-field between 1997 and 2006 were reviewed retrospectively. Biologically effective dose (BED) was calculated for dose homogenization and was corrected with the factor of overall radiation time. Patients were divided into low BED group (n = 70) and high BED group (n = 135) with a cut-off of BED 57 Gy . Outcomes of the two groups were compared.p = 0.024), progression-free survival (p = 0.006) and overall survival (p = 0.005), with a trend toward improved distant-metastasis free survival (p = 0.196). Multivariable Cox regression demonstrated that age (p = 0.003), KPS (p = 0.009), weight loss (p = 0.023), and BED (p = 0.004) were significant predictors of overall survival.Median follow-up was 20.7 months for all analyzable patients and 50.8 months for surviving patients. Considering all patients, median survival was 22.9 months ; 2- and 5-year survival rates were 47.2% and 22.3%, respectively. Patients in high BED group had a significantly better local control (Our data showed that a high BED was significantly associated with favourable outcomes in the Chinese LS-SCLC population, indicating that a positive BED-response relationship still existed even in a relatively high radiation dose range. Although concurrent thoracic radiotherapy (TRT) combined with chemotherapy represents the standard of care in the management of limited-stage small-cell lung cancer (LS-SCLC), the optimal radiation schedule and total dose for LS-SCLC remain topics of continuous debate ,2. In thMultiple studies have confirmed that there is a radiation dose-response for SCLC but the radiation dose evaluated was often in the lower range of 25-50 Gy -12. ChoiAs SCLC presents the biological characteristics of sensitivity to treatment and early spread to distant sites, we really do not know whether further increase of TRT dose is necessary for LS-SCLC. Our concern is whether a dose-response relationship still exists for improved LC and OS in LS-SCLC when a certain threshold of TRT intensity has been reached. Unfortunately, few studies have been specifically addressed this critical issue for LS-SCLC. In order to evaluate if there is a dose-response relationship, the outcome of LS-SCLC patients treated consecutively at our centre with combination of chemotherapy and TRT with doses greater than 50 Gy were reviewed. Since radiation dose confounds both fractionation and overall radiation time (ORT), the biologically effective dose (BED) with ORT will be a more appropriate representative of the biological effect than the single physical dose. Thus we investigated the underlying BED-response relationship for LS-SCLC in this study.Medical and RT records of all patients with LS-SCLC between 1997 and 2006 were reviewed. Patients were selected based on the initial diagnosis of LS-SCLC where definitive TRT with doses equal or greater than 50 Gy was carried out as a part of their treatment for this disease. All patients had histology confirmed SCLC by bronchoscopic, transthoracic biopsy or sputum cytology no less than twice. Pre-treatment staging procedures consistently included clinical history, physical examination, biochemical test, computed tomography (CT) scan of the thorax and abdomen, magnetic resonance imaging or CT scan of the brain, and bone scan. Limited-stage disease was defined as disease confined to one hemithorax which can be safely encompassed within a tolerable radiation field. Presence of an ipsilateral pleural effusion was classified as limited-stage if cytology was negative or if the effusion was small.A total of 234 patients were identified as LS-SCLC in the period, 29 were excluded because they had undergone surgery (n = 14) or had been treated to dose < 50 Gy (n = 15). For all 205 patients with definitive chemoradiotherapy, median age at diagnosis was 62 years (range 35-83) and median KPS was 80 (range 60-100).Treatment strategies were determined on the basis of tumor status, patient's performance and comorbidities at the discretion of the treating oncologist, and referring to the clinical practice guidelines formulated in our centre. The majority of patients were given modified chemoradiotherapy because of concerns of serious toxicity from concurrent chemoradiotherapy and insufficient supportive treatment in developing country . GeneralDuring the period, TRT was delivered with megavoltage equipment (6-15 MV), and either two-dimensional or three-dimensional techniques were allowed. The gross target volume (GTV) was based on the restaging chest CT obtained after the last induction chemotherapy, including the primary tumor (post-chemotherapy) and all clinical/radiological involved lymphatic regions with a short-axis diameter ≥ 1 cm (pre- or post-chemotherapy). Elective treatment of clinically uninvolved lymphatic regions was not carried out. No specific clinical target volume (CTV) was used in this population. A margin of 1.0-1.5 cm was placed to form planning target volume (PTV) according the site and motion of the target (the margin of 1.5 cm was commonly used in the most of patients). Typically, patients with two-dimensional planning were treated with equally weighted AP-PA fields to 40-42 Gy, then boost by parallel opposed off cord oblique fields to the prescribed dose. For patients with three-dimensional planning, three to six coplanar photon fields were used and the prescribed dose was corrected for lung inhomogeneity. As for the dose fractionation scheme, both once-daily and twice-daily fractions were used in the period, which was chosen mainly depended on the attending physician's judgment and preference. For patients with once-daily TRT, a total dose of 50-70 Gy was administered at 1.8-2.5 Gy per fraction. For patients with twice-daily TRT, a total dose of 56 Gy at 1.4 Gy per fraction was delivered at intervals longer than 6 h, in 40 fractions over 4 weeks, which has been described previously .To enable comparison of the physical dose values with different fractionation schemes, we calculated the BED using the linear quadratic formula that included the factor of ORT which could take into account for the accelerated proliferation during irradiation course .k is the delay in proliferation in tumors ('kick-off time' is assumed to 21 days), Tpot is the potential doubling time of the tumor clonogenic cells which is set to 3 days for SCLC [Where n is the number of fractions, d is the fraction size, α/β ratio is 10 Gy, α is 0.3 Gy, T is the ORT considering that the first fraction was given on day 1, Tfor SCLC . In our 3, neutropenic fever or sepsis, and platelet count ≤ 50,000 mm3. Loco-regional symptoms included severe esophagitis , and severe pneumonitis .In this study, toxicity associated with TRT was reported as days of interruption during the course of TRT except for holidays and mechanical failures. The hematologic criteria for interruption included absolute neutrophil count ≤ 1000/mmGenerally, patients were followed up every 3-4 months for 2 years, then every 6 months thereafter. The survival status of patients lost to follow-up was updated with the information from the R.P.C Social Security System. OS was the primary endpoint of this study which was measured from the start date of any treatment to patients' death from any cause or the last follow-up. Only the first treatment failure was taken into account. Progression-free survival (PFS) was defined as the duration of survival without loco-regional recurrence or distant metastases. Local recurrence was defined as disease progression within the irradiated field alone or together with distant metastases , while progression of tumor out-of-field was not included in this analysis, provided that this kind of loco-regional recurrence can't be controlled by TRT intensification. Distant-metastases free survival (DMFS) was defined as the interval from the day that treatment initiated to the day of distant metastases occurred or the last follow-up. All endpoints were estimated by Kaplan-Meier model.p values were two-sided, with p ≤ 0.05 considered significant.The Duke's experiences showed that LS-SCLC patients treated with approximately 60 Gy once-daily TRT have promising outcomes , and thiAt the present analysis, 42 patients (20.5%) were alive, 153 dead (74.6%) and 10 censored (4.9%). Median follow-up time was 20.7 months (range 3.6-102.8 months) for all analyzable patients and 50.8 months (range 27.3-102.8 months) for patients alive. Considering all patients, median OS was 22.9 months ; 2- and 5-year OS were 47.2% and 22.3%, respectively.p = 0.000). Additionally, it should be mentioned that we also evaluated the size of equivalent square field at anterior-posterior axis as an alternative indicator of tumor volume for each patient, considered that the prescribed TRT dose may be affected by the tumor volume. In some cases treated with three-dimensional conformal TRT, a virtual field was utilized to generate the size. As a result, there was no significant difference between the two groups.Of the 205 patients, 70 received BED ≤ 57 Gy (low BED group) and 135 > 57 Gy (high BED group). Table p = 0.005, Figure p = 0.006, Figure The median OS for patients treated with low BED and those with high BED were 16.4 months  and 25.4 months ; 2- and 5-year OS were 31.5% and 14.6%, 55.2% and 26.2%, respectively . Table p = 0.681). Seven patients (3.4%) experienced Grade 3 acute pneumonitis, defined as severe coughing or dyspnea requiring oxygen inhalation. There was no difference between the two groups in the incidence of Grade 3 pneumonitis . A total of 46 patients (22.4%) required treatment interruptions during TRT due to hematologic and/or loco-regional toxicities. The factors directly leading to treatment interruptions were esophagitis (40.4%), neutropenia (29.8%), pneumonitis (12.8%), nausea and vomiting, dehydration, and others (17.0%). The median duration of treatment "break" was 6 days (range 1-18). Thirteen patients (18.6%) in low BED group experienced treatment breaks, while 33 (24.4%) in high BED group did. No statistically significant difference was found in the incidence of interruptions as a function of BED (p = 0.339).The most common acute complication was radiation esophagitis. There was no significant difference between the low and high BED groups in the incidence of Grade 3 esophagitis, defined as an inability to swallow solids, requiring narcotic analgesics or the use of a feeding tube . The time from the start of any treatment to the end of the TRT (SER) was not a significant factor for OS when SER was analyzed as a continuous variable (p = 0.530) or as a categorical variable based on the median value (p = 0.623). There were no significant differences in OS based on sex, lactate dehydrogenase, ipsilateral supraclavicular nodes, daily fraction, TRT technique, chemotherapy cycles or ORT.The effects of patient- and treatment-characteristics on OS are shown in Table p = 0.057). Figure p = 0.012).Multivariate analysis demonstrated that age ≤ 65 years, high KPS, weight loss ≤ 5% and high BED remained significantly correlated with improved OS Table , while PThis retrospective study showed that patients treated with BED > 57 Gy had significantly better LC, PFS and OS in LS-SCLC, indicating that patients could achieve benefits from high BED. This result is consistent with previous findings that TRT dose intensification improved LC, resulting in better outcomes in LS-SCLC ,5,17. SpThe radiation dose ≥ 50 Gy determined as the inclusion criteria for this retrospective analysis was based on our assumption that 50 Gy might be a conservative radiation dose for our LS-SCLC population with curative intent when sequential chemoradiotherapy was given, according to our previous study . In the Accelerated proliferation of tumor clonogens during radiotherapy has been shown to affect outcomes in many malignant solid tumors -22. Two Indeed the biological radiation dose (without time correction) in the high once-daily ,7 or thep = 0.000). However, there was no significant difference in 5-years OS between the once-daily and twice-daily groups, . This indicated that high BED administered in once-daily scheme might lead to non-inferior outcomes compared with twice-daily scheme for LS-SCLC. Therefore, we believed that the difference of daily fraction scheme between the two groups had no apparent impact on our conclusions.The distributions of patient- and treatment-related characteristics were similar for low and high BED groups except for daily fraction scheme. Twice-daily scheme was more frequent in high BED group than in low BED group  plus concurrent chemotherapy in patients with LS-SCLC is ongoing in our centre, with the hypothesis that both high TRT dose and short ORT are important for the treatment of LS-SCLC.The authors declare that they have no competing interests.BX and XLF designed this study, performed much of the work, and drafted the manuscript. Patient accrual and clinical data collection was done by all authors. XWC and JDZ participated in the analysis and the data interpretation. All authors read and approved the final manuscript.


It is generally believed that incretin-based therapies are effective in patients possessing certain levels of preserved β-cell function. So far, there are no reports that show the effectiveness of dipeptidyl peptidase-4 inhibitors in patients who absolutely lack the capacity for endogenous insulin secretion.This report describes the efficacy of sitagliptin in three Japanese patients  who had no detectable post-meal C-peptide levels. Although they were receiving intensive insulin therapy together with some oral hypoglycemic agents, their glycemic control remained poor. Sitagliptin was added to the ongoing therapeutic regimen to provide better glycemic control. Although these patients had mild hypoglycemia, effective reductions of hemoglobin A1c levels were observed without any adverse events in the liver and kidney during the following 24 weeks. Two of the patients were able to reduce their insulin doses, and one of the patients could discontinue one of the oral hypoglycemic agents. There was no weight gain or gastrointestinal complaints among the three patients. Post-meal C-peptide levels remained undetectable after sitagliptin treatment.This report demonstrates that sitagliptin is effective and safe as an add-on therapy to insulin in reducing blood glucose levels in patients who absolutely lack the capacity for endogenous insulin secretion. The improvement seen in glycemic control could not be due to enhanced endogenous insulin secretion, since post-meal C-peptide levels remained undetectable after sitagliptin treatment, but it could be a result of other factors . However, the glucagon-suppressive effect of sitagliptin is known to be rather weak and short-lived. Given this background, a novel hypothesis that the glycemic effects of this drug may be caused by mechanisms that are independent of the glucagon-like peptide 1 axis (extra-pancreatic effect) will be discussed. The incretin-based drugs glucagon-like peptide 1 (GLP-1) receptor agonists and dipeptidal peptidase 4 (DPP-4) inhibitors are a new class of drugs for the treatment of type 2 diabetes . CurrentCase 1 is a 91-year-old Japanese woman who had been diagnosed with acute onset of type 1 diabetes two years earlier and had been using various doses and types of insulin since that diagnosis. Her endogenous insulin secretory capacity was almost completely lost since her post-meal  C-peptide levels were undetectable (below 0.1 ng/ml) and 24-hour urine C-peptide levels were below 1.1 mg/day . Her insulin antibodies were positive at diagnosis. Why this patient developed acute onset of autoimmune type 1 diabetes with absolute insulin deficiency is elusive.This may be an atypical case. Her post-meal glucagon level was 130 pg/ml . Her body mass index (BMI) was 21.5 (body weight/body height = 40.4 kg/137 cm). Her hemoglobin A1c (HbA1c) level was above 9.5% in the three months prior to this study. Sitagliptin (25 mg/day) was added to the ongoing therapeutic regimen to provide better control of her diabetes. At that time, she was using premixed biphasic insulin aspart  three times daily before each meal . She was also taking 50 mg of miglitol three times daily and 150 mg/day aliskiren. Her post-meal blood glucose was 252 mg/dl, and her HbA1c level was 9.6% at the start of sitagliptin therapy (at baseline). She had occasionally mild hypoglycemia after starting sitagliptin, which she could manage by taking glucose drinks by herself. Her HbA1c levels were followed for 24 weeks and were reduced from 9.6% to 7.4% (2.2% reduction)  and insulin antibodies were negative (below detectable range). Therefore, this patient has features of both type 1 and type 2 diabetes. However, because of his clinical picture, this patient may have non-autoimmune fulminant type 1 diabetes, which is rare in Caucasian populations but comprises approximately 10% of type 1 diabetes cases in Japan . His endCase 3 is a 54-year-old Japanese man who had been diagnosed with type 2 diabetes 22 years earlier and had been using different types and doses of insulin since that diagnosis. His endogenous insulin secretory capacity was almost completely lost, since his post-meal C-peptide levels were repeatedly undetectable below 0.1 ng/ml). His GAD and insulin antibodies were negative. His BMI was 22.9 (body weight/body height = 64 kg/167 cm). His HbA1c level was above 7.5% in the three months prior to this study. Sitagliptin (50 mg/day) was added to the patient's therapeutic regimen to try to achieve better control of his diabetes. At that time, he was using insulin lispro  three times daily before each meal  and insulin glargine  before going to bed (20U) and was also taking 2.5 mg/day rosuvastatin, 250 mg metformin three times daily, 5 mg/day amlodipine and 2 mg/day doxazosin mesylate. His post-meal blood glucose level was 221 mg/dl, and his HbA1c level was 7.5% at baseline. He had occasionally mild hypoglycemia after starting sitagliptin, which he could manage by taking glucose drinks by himself. His HbA1c level was followed for 24 weeks and was reduced from 7.5% to 6.5% (1.0% reduction) . One of the features of such patients is that they lack the capacity for endogenous insulin secretion. Increasing the dosage of insulin units may be a solution to this problem; however, in practice, this can cause an increased risk of hypoglycemia and weight gain. Incretin-based drugs (GLP-1 receptor agonists and DPP-4 inhibitors) may help such patients attain desirable glycemic control. Furthermore, these drugs induce benefits in terms of post-prandial hyperglycemia control in addition to positive effects on β-cell function and possible β-cell preservation .Recently, sitagliptin, the first DPP-4 inhibitor launched on the market, has been approved for use in combination with insulin. Sitagliptin combined with insulin significantly reduces blood glucose levels over the four-hour period following the meal test. Serum C-peptide and insulin levels were found to significantly increase over the four-hour period following the meal with sitagliptin in comparison to that in patients receiving a placebo ,9. In thSo, what are the potential mechanisms that can cause the reduced glucose levels with sitagliptin? A number of explanations can be postulated.First, the glycemic effect observed in these patients may be due to suppression of glucagon levels. The mechanism by which GLP-1 suppresses glucagon secretion is far from being elucidated, and furthermore it is unclear whether endogenous insulin is a required factor for this effect. On the basis of the data from the present case report and others ,9, one mSecond, the improvement seen in glycemic control with sitagliptin therapy could be due to the pharmacological effects of GLP-1, such as delayed gastric emptying and body weight loss. However, this does not seem to be the case, since in contrast to GLP-1 analogues, DPP-4 inhibitors, including sitagliptin, do not have any clear effects on these parameters . The patFinally, the background information described above might suggest that the glucose-lowering effect of sitagliptin not only occurs via the GLP-1 axis but is also mediated by novel mechanisms including extra-pancreatic signal transduction pathways. Specifically, sitagliptin may have an independent role in the regulation of hepatic glucose output, glycogenesis, gluconeogenesis, glycolysis or peripheral  glucose uptake different from that of insulin or glucagon action. Alternatively, it may have a novel glycemic effect, such as inhibiting glucose absorption by the intestine. These putative effects are illustrated in Figure The strength of this work is that similar results were obtained from patients who have very different diabetic backgrounds. But the common denominator is that these patients are absolutely insulin-deficient. A limitation of this work is that this report is based on just three observational case studies. Well-designed and adequately powered randomized clinical trials are necessary to draw well-validated conclusions.The cases presented in this report demonstrate that sitagliptin is still effective and safe in reducing blood glucose levels in those patients who have no residual β-cell function. However, one should keep in mind that sitagliptin should be used not as a replacement therapy but as an add-on to insulin with such absolutely insulin-deficient patients.BMI: body mass index; DPP-4: dipeptidyl-peptidase-4; GAD: glutamic acid decarboxylase; GLP-1: glucagon-like peptide 1.Written informed consent was obtained from the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.EK analyzed and interpreted the patient data and wrote the manuscript.


In accordance, Ex4 also blocks nicotine-induced expression of locomotor sensitization in mice. Given that development of nicotine addiction largely depends on the effects of nicotine on the mesolimbic dopamine system these findings indicate that the GLP-1 receptor may be a potential target for the development of novel treatment strategies for nicotine cessations in humans. The gastrointestinal peptide glucagon-like peptide 1 (GLP-1) is known to regulate consummatory behavior and is released in response to nutrient ingestion. Analogues of this peptide recently emerged as novel pharmacotherapies for treatment of type II diabetes since they reduce gastric emptying, glucagon secretion as well as enhance glucose-dependent insulin secretion. The findings that GLP-1 targets reward related areas including mesolimbic dopamine areas indicate that the physiological role of GLP-1 extends beyond food intake and glucose homeostasis control to include reward regulation. The present series of experiments was therefore designed to investigate the effects of the GLP-1 receptor agonist, Exendin-4 (Ex4), on established nicotine-induced effects on the mesolimbic dopamine system in mice. Specifically, we show that treatment with Ex4, at a dose with no effect  Nicotine addiction, a major health problem, cause a wide range of serious effects such as cancer, chronic obstructive pulmonary disease and cardiovascular disease . DevelopThe gut-brain hormone glucagon-like peptide 1 is secreted in response to nutrient ingestion  from entad libitum, except during the experimental setups. The study was carried out in strict accordance with the recommendations in the Swedish Animal Welfare Act and all experiments were approved by The Swedish Ethical Committee on Animal Research in Gothenburg. All efforts were made to minimize animal suffering, and to reduce the number of animals used. All animals were allowed to acclimatize at least one week before the start of the experiments.Adult post-pubertal age-matched male NMRI mice  were used. All mice were group housed and maintained at a 12/12 hour light/dark cycle (lights on at seven am). Tap water and food  were supplied per se and this dose blocked the rewarding properties of alcohol in mice [Nicotine ditartrate  was dissolved in vehicle (0.9% sodium chloride solution), and sodium bicarbonate was added until the pH was natural. The selected dose of nicotine, 0.5 mg/kg, was based on a previous dose response study where this dose increased the locomotor activity in mice . Nicotin in mice . The selLocomotor activity was recorded as described previously . In brieper se [The mice were allowed to habituate to the locomotor activity box one hour prior to drug challenge. The effects of Ex4  on nicotine-induced  locomotor stimulation were investigated. This dose of Ex4 was previously determined as the highest dose with no effect per se . Ex4 wasFor measurements of extracellular dopamine levels, mice were implanted unilaterally with a microdialysis probe positioned in the nucleus accumbens. The surgery was conducted two days prior to measurements of dopamine and was performed as described in detail previously . In brieThe effect of systemic administration of Ex4  on nicotine-induced  accumbal dopamine release was investigated using microdialysis in freely moving mice. On the day of the experiment the probe was connected to a microperfusion pump  and perfused with Ringer solution at a rate of 1.5 μl/minute. After one hour of habituation to the microdialysis set-up, perfusion samples were collected every 20 minutes. The baseline dopamine level was defined as the average of three consecutive samples before the first drug/vehicle challenge, and the increase in accumbal dopamine was calculated as the percent increase from baseline. After the baseline samples (-40 minutes until 0 minutes), mice were injected with Ex4 or vehicle (at 5 minutes), which was followed by a nicotine or vehicle injection (at 20 minutes). Following these drug administrations an additional eight 20 minute samples were collected. The following treatment groups (n=8 in each group) were tested: vehicle-vehicle (Veh-Veh), vehicle-nicotine (Veh-Nic), Ex4-vehicle (Ex4-Veh) and Ex4-nicotine (Ex4-Nic) (n=8 in each group).The dopamine levels in the dialysates were determined by HPLC with electrochemical detection. A pump , an ion exchange column  and a detector  equipped with a VT-03 flow cell (Antec Leyden) were used. The mobile phase (pH 5.6), consisting of sulfonic acid 10 mM, citric acid 200 mM, sodium citrate 200 mM, 10% EDTA, 30% MeOH, was vacuum filtered using a 0.2 μm membrane filter . The mobile phase was delivered at a flow rate of 0.2 ml/minute passing a degasser , and the analyte was oxidized at +0.4 V.After the microdialysis experiments were completed, the mice were decapitated, and probes were perfused with pontamine sky blue 6BX to facilitate probe localization. The brains were mounted on a vibroslice device  and cut in 50 μm sections. The location of the probe was determined by gross observation using light microscopy. The exact position of the probe was verified  and onlyper se [To evaluate the effects of Ex4 on the rewarding effects of nicotine, CPP tests were performed in mice as previously described ,25. In bper se . Conditioned place preference was calculated as the difference in % of total time spent in the drug-paired (i.e. least preferred) compartment during the post-conditioning and the pre-conditioning session. The present experiment was designed to reflect CPP expression rather than acquisition of CPP. Interestingly, it has been suggested that pharmacological agents that are administered only on the test day, as in the present design, should be considered as being potential candidates in the treatment of human drug craving (for review see ).Repeated administration of addictive drugs causes an increased locomotor response , i.e. a Locomotor activity data were evaluated by a one-way ANOVA followed by Bonferroni post-hoc tests. The microdialysis experiments were evaluated by a two-way ANOVA followed by Bonferroni post-hoc test for comparisons between different treatments and specifically at given time points. The condition place preference data were evaluated by an unpaired t-test. The locomotor sensitization data were analyzed with a two-way ANOVA followed by Bonferroni post-hoc tests. Data are presented as mean ± SEM. A probability value of P<0.05 was considered as statistically significant.P=0.0005; n=8 per group). As shown in P<0.001). This nicotine-induced locomotor activity was significantly reduced by pre-treatment with a single injection of Ex4 (P<0.01), at a dose that alone had no significant effect on locomotor activity compared to vehicle treatment (P>0.05). There was no difference in locomotor activity response in vehicle treated mice and Ex4-Nic treated mice (P>0.05).An overall main effect of treatment on locomotor activity was found in mice following systemic administration of nicotine (0.5 m g/kg) and Ex4 (2.4 μg/kg) =8.11, P>0.05). There was no difference in accumbal dopamine response in vehicle treated mice and Ex4-Nic treated mice at any time interval (P>0.05). Only mice with correct probe placement in the nucleus accumbens shell were included in the statistical analysis =61.15, P<0.0001) and treatment x time interaction =3.45, P<0.0001), but not of time F=1.28, P>0.05)(n=8 in each group). As shown in analysis . P<0.05, n=8 in each group; The nicotine-induced (0.5 mg/kg) (Nic-Veh) expression of CPP was significantly attenuated by an acute single injection of Ex4 (2.4 μg/kg) (Nic-Ex4) on the post-conditioning day compared to vehicle injection (P<0.0001), time F=20.37, P=0.0005) as well as treatment x time interaction =36.75, P=0.0008)(n=8 in each group). The locomotor sensitization data revealed an overall main effect of treatment =10.57, P<0.001). Acute injection with Ex4 on the last day (i.e. day 8) blocked this nicotine-induced locomotor sensitization ((P>0.05) (Nic-Veh vs Nic-Ex4). The selected dose of Ex4 had no effect on locomotor activity per se (P>0.05) (Veh-Veh vs Veh-Ex4).. As shown in The present study provides novel evidence showing that GLP-1 receptors regulate nicotine-induced activation of the mesolimbic dopamine system in mice, suggesting that the physiological role of GLP-1 extends beyond control of glucose homeostasis and food intake ,11. IndeGlucagon-like peptide 1 containing-fibers from the NTS target reward areas expressing GLP-1 receptors such as the VTA and N.Acc ,9,17,30.Our present findings show for the first time an involvement of the GLP-1 receptor in nicotine-induced activation of the mesolimbic dopamine system as measured by locomotor stimulation, accumbal dopamine release and expression of CPP. In addition we showed that Ex4 blocks the nicotine-induced expression of locomotor sensitization in mice. Given that expression of locomotor sensitization has been suggested to reflect the neurochemical alterations underlying important parts of addiction such as craving and compulsive drug taking , GLP-1 rAlbeit previous studies have shown that Ex4, at a dose range similar to what was used in the present study, induces a condition taste aversion  the possThe present data support the hypothesis that common mechanisms, such as gut-brain hormones, regulate both food- and drug-induced activation of the mesolimbic dopamine system . Previou